Process for producing fermentation product

ABSTRACT

A process for producing a fermentation product by effecting fermentation with the use of a bioreactor having an immobilized microorganism located therein, characterized in that a non-flocculent yeast is employed as the microorganism.

TECHNICAL FIELD

The present invention relates to a method of making a fermentationproduct; and, more specifically, to a method of making a fermentationproduct by carrying out fermentation by use of a bioreactor within whichan immobilized microorganism is disposed.

BACKGROUND ART

As biotechnology advances, the making of fermentation products by use ofbioreactors utilizing immobilized microorganisms is under study in thefields of brewing for malt alcohol beverages (beers), wines, sake,vinegar, soy sauce, and the like. When a bioreactor is used as such, thefollowing are expected:

1) Since a high concentration of yeast is immobilized to carry outfermentation, the brewing is completed rapidly, so that the brewingperiod can be shortened, whereby the number of manufacturing tanks andthe cost of construction can be lowered.

2) Since continuous fermentation is possible, it is unnecessary tocharge and collect yeast.

Conventionally, when a beer or the like is manufactured by use of abioreactor, however, the amount of amino acids and diacetyl (DA) hasbecome greater, and the amount of ester has become smaller in theproduct as compared with a product manufactured by a traditional batchfermentation method. As a result, the product obtained by use of thebioreactor has been problematic in that it is disadvantageous in flavor,and this problem has been remarkable in particular when the bioreactoris used for primary fermentation (main fermentation).

For overcoming such a problem, studies have conventionally been carriedout. Japanese Patent Application Laid-Open Gazette No. HEI 7-123969discloses a method in which a fermentation liquid is circulated in acontinuous fermentation method using a fluidized bed type reactor.Though the consumption of amino acids in the fermentation process isameliorated by this method, the amount of diacetyl which causes raw odoror immature odor is not lowered sufficiently, whereby the resultingproduct still has room for improvement in terms of flavor. Also,manufacturing methods in bioreactors including this method have beenproblematic in that the number of floating yeast cells upon the end(completion) of primary fermentation is small in general, thefermentation rate in the process of fermentation is unstable, and theyare hard to control.

Meanwhile, it has conventionally been known that yeast for making afermentation product such as beer is required to have flocculationability (agglutinability) to a certain extent. Here, flocculationability refers to a property in which yeast cells flocculate as a massupon the end of fermentation and are separated from the liquid so as tofloat up or sediment. If the flocculation ability is too high, then theflocculation occurs at an earlier stage, so that the contact between thefermentation liquid and yeast is broken, whereby fermentation becomesinsufficient. If the flocculation ability is too low, by contrast, thenyeast floats in the liquid for a long time, so that the amount ofrecovery of yeast decreases, which takes unnecessary time and labor forseparating and collecting yeast by centrifuge or the like, for example.Therefore, it has conventionally been common knowledge to use yeasthaving a certain degree of flocculation ability (flocculent yeast) formaking a fermentation product. Such flocculent yeast (agglutinativeyeast) is used in the above-mentioned method disclosed in JapanesePatent Application Laid-Open Gazette No. HEI 7-123969 as well.

In view of the above-mentioned problems in the prior art, it is anobject of the present invention to provide a method in which, whenmaking a fermentation product by use of a bioreactor utilizing animmobilized microorganism, the fermentation rate in the fermentationprocess can be held constant, and the number of floating yeast cellsupon the end of fermentation can stably be maintained at a level higherthan the conventional one, whereby, when making a malt alcohol beverageby use of a bioreactor in particular, the amount of diacetyl in thefermentation liquid and final product can be lowered sufficiently and soforth, so as to improve the flavor of final product.

DISCLOSURE OF THE INVENTION

The inventors have repeated diligent studies in order to achieve theabove-mentioned object, and have found that non-flocculent yeast(nonagglutinative yeast) can be employed contrary to the conventionalknowledge when using a bioreactor utilizing an immobilizedmicroorganism, since it is not necessary to separate and collect yeast,so that the fermentation rate in the fermentation process becomesconstant, the number of floating yeast cells upon the end offermentation is stably maintained at a level further preferable forsecondary fermentation (after-fermentation), the amount of diacetyl inthe fermentation liquid and final product sufficiently decreases in thecase using such a bioreactor for primary fermentation of a malt alcoholbeverage in particular, and so forth, whereby the flavor of productimproves. Thus, the present invention has been accomplished.

Namely, the method of making a fermentation product in accordance withthe present invention is a method of making a fermentation product byusing a bioreactor within which an immobilized microorganism isdisposed, wherein non-flocculent yeast is used as the microorganism.

The malt alcohol beverage of the present invention is made by the methodof the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an example of fluidized bed typereactor suitable for the present invention;

FIG. 2 is a graph showing relationships between the batch number offermentation times (the number of continuous fermentation processes) andthe number of floating yeast cells;

FIG. 3 is a graph showing relationships between the batch number offermentation times and the amount of extract consumption;

FIG. 4 is a graph showing relationships between the batch number offermentation times and the number of floating yeast cells;

FIG. 5 is a graph showing relationships between the batch number offermentation times and the amount of diacetyl;

FIG. 6 is a graph showing a relationship between the batch number offermentation times and the amount of extract consumption;

FIG. 7 is a graph showing a relationship between the batch number offermentation times and the number of floating yeast cells; and

FIG. 8 is a graph showing a relationship between the batch number offermentation times and the amount of diacetyl.

BEST MODES FOR CARRYING OUT THE INVENTION

In the following, preferred embodiments of the present invention will beexplained in detail.

The present invention is a method of making a fermentation product bycarrying out fermentation by using a bioreactor within which animmobilized microorganism is disposed, wherein non-flocculent yeast isused as the microorganism.

First, the yeast and its immobilizing carrier in accordance with thepresent invention will be explained. Any yeast can be used in thepresent invention as long as it is non-flocculent as will be explainedlater, and non-flocculent ones are selected from yeast speciescorresponding to the aimed fermentation product. For example, as yeastused for making a liquor, non-flocculent ones are selected fromso-called liquor yeast species which produce alcohol, carbonic acid gas,and the like by metabolizing a brewing material liquid. Specifically,non-flocculent ones are selected from Saccharomyces cerevisiae,Saccharomyces uvarum, and the like. Examples of such liquor yeastinclude non-flocculent beer yeast, non-flocculent wine yeast, andnon-flocculent sake yeast. For example, non-flocculent beer yeast may beused so as to make a malt alcohol beverage such as beer.

Here, flocculation ability refers to a property in which yeast cellsdispersed in a fermentation process flocculate (aggregate) so as toattach and bind to each other at cell surfaces, thereby forming a floc.Bottom fermentation yeast species used for normal beer productioninclude strains which tend to be rapidly separable from within afermentation liquid by flocculating (aggregating) their cells to form afloc, and strains which are hard to flocculate and are likely todisperse and float for a relatively long period of time. The former arereferred to as “flocculent yeast,” whereas the latter are referred to as“non-flocculent yeast (also known as dust-like yeast).”

While it has been reported that such flocculation ability of yeast isessentially a genetic characteristic of yeast itself and is controlledby Lg-FLO 1 gene existing at a position corresponding to the VIIIchromosome of Saccharomyces cerevisiae (Japanese Patent ApplicationLaid-Open Gazette No. HEI 8-266287), it may also be influenced by waterfor brewing, materials, malt compositions, malt aeration conditions,yeast culture conditions, fermentation vessels, handling of yeast, andthe like, and the strength of flocculation ability may vary as thefermentation process proceeds. Thus, the strength of flocculationability varies depending on yeast strains, and the same species of yeastmay include non-flocculent and flocculent ones depending on strains.Therefore, it is preferred in the present invention that anon-flocculent strain be selected by the following method and the like,so as to use non-flocculent yeast derived from this strain. Inparticular, it is preferred to use such non-flocculent yeast alone.

An example of methods for measuring the flocculation ability of yeast soas to select non-flocculent yeast to be used for the method of thepresent invention as such is one described in YEAST GENETICS:FUNDAMENTAL AND APPLIED ASPECTS, 205–224 (1983). Specifically, thefollowing method is preferred.

Namely, 0.6 g of yeast (one separated by precipitation upon centrifugeat 3000×g for 10 minutes at the time when fermentation is completed) isadded to 20 ml of tap water, so as to yield a yeast suspension. To 9 mlof this yeast suspension, 1 ml of 0.5-M acetic acid buffer solution atpH 4.5 including 1500 ppm of calcium ion is added. The resulting mixtureis hand-shaken up and down, for example, so as to uniformly stir it as awhole. Then, the mixture is left to stand still for 5 minutes at roomtemperature.

Thereafter, the degree of agglutination is visually inspected (by thenaked eye), and is evaluated according to the following four criteria:

0: Non-flocculent (No boundary is seen between the liquid and floc bythe naked eye, and neither flocculation (aggregation) nor sedimentationof yeast is observed.)

1: Weakly Flocculent (Though no boundary can be seen between the liquidand floc by the naked eye, flocculation or sedimentation of a part ofyeast is observed.)

2: Mildly Flocculent (A boundary can be seen between the liquid and flocby naked eye, and flocculation or sedimentation of yeast is observed.)

3: Strongly Flocculent (Yeast substantially completely flocculates andsediments, and the supernatant becomes substantially completely clear.)

It is preferred in the present invention that a non-flocculent strainsatisfying the non-flocculent condition (level 0) in the above-mentionedevaluation criteria be selected, and non-flocculent yeast derived fromthis strain be used. In particular, it is preferable to use suchnon-flocculent yeast alone.

Thus, contrary to the conventional knowledge, the method of the presentinvention uses non-flocculent yeast derived from the non-flocculentstrain, so that yeast is fully prevented from flocculating within thebioreactor in the process of fermentation and thereby sedimenting andprecipitating, whereby the fermentation rate is held constant, and thenumber of floating yeast cells upon the end of fermentation is stablymaintained at a level higher than that in the case where conventionalflocculent yeast is used. In the case where the method of the presentinvention is employed in primary fermentation of a malt alcohol beveragein particular, the amount of diacetyl in the fermentation liquid uponthe end of primary fermentation is sufficiently lowered since the numberof floating yeast cells upon the end of primary fermentation improves,and diacetyl is further efficiently reduced in secondary fermentation,so that the amount of diacetyl in the final product sufficientlydecreases, thus lowering the resulting immature odor component, wherebythe flavor of product improves.

Without being restricted in particular, various kinds of carriers can beused as a carrier for immobilizing the above-mentioned non-flocculentyeast. In particular, carriers comprising chitin-chitosan, alginic acid,carrageenan are preferable. Among others, chitosan type beads (carriersmade from chitin-chitosan obtained by actylation of chitosan) arepreferable. Since chitosan type beads are hydrophilic and porous,carbonic acid gas is easily let out therefrom. Also, they are hard towear, and their fluidity is favorable since their density approximatesthat of the material liquid. Further, the chitosan type beads can hold alarge amount of microorganisms, thereby tending to shorten thefermentation time more. Also, since microorganisms are adsorbed andimmobilized by the chitosan type beads relatively mildly, their growthand desorption become easier, and dead cells are kept from continuouslyexisting within the carrier as in a collective carrier.

Any method can be used for the sterilization carried out beforeimmobilizing non-flocculent yeast onto such a carrier. Preferred arehigh-pressure sterilization method, sterilization method using causticsoda, sterilization method using steam, and the like. Also, the methodof immobilizing non-flocculent yeast onto the carrier is not restrictedin particular. While its examples include one in which the carrier isadded to a yeast suspension, and then the mixture is stirred or theliquid is circulated, other known methods may be used as well.

The bioreactor in accordance with the present invention will now beexplained. The bioreactor in accordance with the present invention isone within which the immobilized microorganism (non-flocculent yeastimmobilized to the carrier) is disposed, and the material liquid andimmobilized microorganism come into contact with each other, wherebyfermentation is carried out. Examples of such a bioreactor include, interms of their types, complete mixed vessel type reactors, packed bedtype reactors, film type reactors, fluidized bed type reactors, andlateral reactors. For fermentation in which alcohol and carbonic acidgas are generated upon metabolizing a brewing material, such as primaryfermentation of malt alcohol beverages, it is preferable to usefluidized bed type reactors which easily let the gas out of the system.

Preferably, such a fluidized bed type reactor comprises a fluidized bedsection within which an immobilized microorganism is disposed, and aliquid circulating section for extracting a part of the fermentationliquid from the downstream side of the fluidized bed section andreturning it to the upstream side of the fluidized bed section. FIG. 1is a schematic diagram showing an example of the fluidized bed typereactor suitable for the present invention.

The fluidized bed type reactor 1 shown in FIG. 1 comprises a reactiontank 2 and a liquid circulating section 3. The reaction tank 2 isconstituted, successively from the upstream side thereof, by arectifying section 4, a fluidized bed section 5, and an empty tubesection 6, whereas the downstream end portion of the reaction tank 2 isprovided with a gas outlet section 7. The liquid circulating section 3is constituted by a pipe 31 connected to the downstream side of thefluidized bed section 5 and to the upstream side of the fluidized bedsection 5 (to the rectifying section 4 in FIG. 1), a pump 32, and avalve 33. Further, the pipe 31 branches off on its way, so as to connectwith a product tank 8 by way of a valve 34. Also, a material liquid tank9 is connected to the upstream end portion of the reaction tank 2 by wayof a pipe 92 having a pump 91.

The fluidized bed section 5 is a section within which the carrier havingimmobilized the microorganism is disposed, whereas the liquidcirculating section 3 is a section for extracting from the downstreamside of the fluidized bed section 5 a part of the fermentation liquid(material liquid) supplied to the reaction tank 2 and returning it tothe upstream side of the fluidized bed section 5 of the reaction tank 2.In the reactor 1 shown in FIG. 1, the extracted fermentation liquid(material liquid) is returned into the reaction tank 2 from therectifying section 4, where the flow of introduced liquid is rectified.The empty tube section 6 is a section where the carbonic acid gas andfermentation liquid generated during fermentation are separated fromeach other as a gas and a liquid; and specifically is the part from theliquid surface to the gas outlet section 7 in the upper portion of thereaction tank 2. The gas such as carbonic acid gas isolated in the emptytube section 6 is let out of the vessel by way of the gas outlet section7.

In the fluidized bed type reactor 1 shown in FIG. 1, the carrier havingimmobilized the microorganism is disposed in the fluidized bed section5, and the material liquid is supplied to the rectifying section 4 fromthe material liquid tank 9 by use of the pump 91, so as to carry outfermentation. Then, a part of the fermentation liquid (material liquid)is extracted from the downstream side of the fluidized bed section 5 byuse of the pump 32 of liquid circulating section 3 and is returned tothe upstream side of the fluidized bed section 5 (to the rectifyingsection 4 in FIG. 4) in the reaction tank 2, whereby fermentation iscarried out while forming a fluidized bed. The carbonic acid gasgenerated during fermentation is let out of the vessel via the gasoutlet section 7 by way of the empty tube section 6 without stayingwithin the fluidized bed, since the fermentation liquid is circulatedand so forth. While the liquid space velocity (linear velocity of fluidper unit volume of fluidized bed) can be changed depending on thedensity of the carrier having immobilized the microorganism, it ispreferably 1 to 20 cm/min, more preferably 1 to 12 cm/min.

It is preferred in the fluidized bed type reactor 1 shown in FIG. 1that, secondary fermentation is completed, the fermented liquidincluding a fermentation product be taken out from the reactor 1 intothe product tank 8 by way of the pump 32 and valve 34, and a newmaterial liquid be supplied from the material liquid tank 9 to thereactor 1 by use of the pump 91, so as to repeatedly carry out thefermentation. Namely, after the end of fermentation, the circulation isstopped and, concurrently with or immediately after the fermented liquidis extracted out of the reactor 1, a new material liquid is supplied tothe reactor 1, so as to repeatedly carry out the fermentation.

While the reactor 1 can be operated by any of batch type, repeated batchtype, and continuous type methods, repeated batch type fermentation ispreferable for yielding a product having a better flavor in a shorttime. The repeated batch type operation tends to yield a better flavorsince the microorganism is grown and renewed during fermentation, whilethe physiological state and growth period of microorganic cells, themicroorganic cell distribution within the reactor 1, and the like aresimilar to those in the batch type operation, which is a traditionalmethod of making liquors.

Any material for making a fermentation product may be used as long as itis suitable for fermentation caused by the nonagglutinative yeastemployed, and known materials can be used at will. For example, malts,fruit juices, sugar liquids, cereal saccharified liquids, and the likeare normally used alone or in combination as appropriate in the makingof liquors. Also, appropriate nutrients and the like may be addedthereto when necessary.

Fermentation conditions are not different from known conditions inessence. For example, the fermentation temperature is normally 15° C. orlower, preferably 8 to 10° C., in the case of brewing malt alcoholbeverages; whereas it is normally 20° C. or lower, preferably 15 to 20°C., in the case of brewing wines.

Examples of fermentation products which can be made by the method of thepresent invention include products in various brewing fields such asmalt alcohol beverages, wines, sake, vinegar, and soy sauce, among whichliquors such as malt alcohol beverages, wines, and sake are preferred.In particular, malt alcohol beverages such as beer are preferable sincetheir flavor is improved by the present invention.

EXAMPLES

In the following, the contents of the present invention will beexplained in more detail with reference to Examples and ComparativeExamples. However, the present invention is not restricted by theseExamples at all.

In the following Examples and Comparative Examples, flocculent strains(A-1, A-2) and non-flocculent strains (NA-1, NA-2, NA-3, NA-4, NA-5)selected, according to the above-mentioned methods and criteria, frombeer yeast (Saccharomyces cervisiae or the like) actually used in thesite of manufacture were used. Table 1 shows the results of evaluationof their flocculation ability. For reference, the results of similarevaluation of flocculation ability concerning type cultures NCYC-No. 203and NCYC-No. 985 in NCYC (National Collection of Yeast Cultures (UnitedKingdom)) are also shown in Table 1.

TABLE 1 RESULT OF FLOCCULATION ABILITY EVALUATION FOR BEER YEAST YEASTRESULT STRAINS USED IN THIS TEST A-1 2 A-2 2 NA-1 0 NA-2 0 NA-3 0 NA-4 0NA-5 0 TYPE CULTURE NCYC-No. 203 2 NCYC-No. 985 0

Examples 1 and 2 and Comparative Example 1

Using the fluidized bed type bioreactor (total volume: 180 ml) shown inFIG. 1, the primary fermentation was carried out by the above-mentionedrepeated batch fermentation (repeated batch type) under the followingconditions. Then, thus obtained fermentation liquid was subjected to afloating yeast cell counting test which will be explained later.

-   -   Primary Fermentation Conditions    -   Carrier: chitosan type beads (Chitopearl HP manufactured by Fuji        Spinning Co., Ltd.)    -   Scale: 50 ml of carrier, 100 ml of wort    -   Strains Used: A-1 (Comparative Example 1), NA-1 (Example 1),        NA-2 (Example 2)    -   Yeast Immobilizing Method: Yeast is immobilized as being brought        into contact with the carrier for 2 days while being circulated        according to a conventional method (2.4 g of sludge yeast with        respect to 50 ml of carrier).    -   Flow Rate: about 1 ml/min    -   Fermentation temperature: constantly 8° C.    -   Fermentation Time: 48 hours/batch    -   Batch number of Fermentation times: 10    -   Wort Used: wort (malt) adjusted to yield a sugar index of 11%        Plato.

Floating Yeast Cell Counting Test

After the end of each primary fermentation process, the number offloating yeast cells in the fermentation liquid was counted by use of aThoma blood cell counting plate. The obtained results are shown in Table2 and FIG. 2. Here, first several repeated fermentation processes wereexcluded from the counting since they correspond to the acclimatizationperiod of yeast in the carrier (ditto in the following).

As can be seen from the results shown in Table 2 and FIG. 2, whennon-flocculent strains were used under the above-mentioned conditions,the number of floating yeast cells upon the end of fermentation wasstably held at 20 to 40 million cells/ml, which was high and closer tothat in normal fermentation. When the flocculent strain was used, bycontrast, yeast was seen to sediment onto the upper part of carrier inthe last half of fermentation, and the number of floating yeast cellsupon the end of fermentation was 10 million cells/ml or less until theseventh fermentation process.

Here, when the number of yeast cells in the carrier was measured after10 fermentation times, it was about 10⁹ cells/1 ml of carrier in each ofthe three strains, whereby it was verified that the number ofimmobilized yeast cells tolerable for practical use in a primaryfermentation bioreactor was achieved in the non-flocculent strains as inthe flocculent strain. When the state of yeast immobilized to thecarrier was observed with an electronic microscope, it was verified thatthe non-flocculent strains were sufficiently immobilized in the chitosantype beads as with the flocculent strain. When the ratio of dead cellsin and out of the carrier was investigated after 10 fermentation times,it was 10% or less in each of the three strains, whereby there was noproblem.

TABLE 2 NUMBER OF FLOATING YEAST CELLS UPON THE END OF PRIMARYFERMENTATION (UNIT: × 10⁶ cells/ml) NUMBER OF FERMENTATION TIMES 1 2 3 45 6 7 AVERAGE NA-1 35 32 33 44 30 36 42 36.0 NA-2 26 32 32 34 14 25 2226.4 A-1 5 7 9 5 8 7 11 7.4

Examples 3 and 4 and Comparative Example 2

Using the fluidized bed type bioreactor (total volume: 20 liters) shownin FIG. 1, the primary fermentation process was carried out by theabove-mentioned repeated batch fermentation (repeated batch type) underthe following conditions. Then, thus obtained fermentation liquid wassubjected to a fermentative property test, a floating yeast cellcounting test, and a diacetyl generated amount test which will beexplained later.

Primary Fermentation Conditions

-   -   Carrier: chitosan type beads (Chitopearl HP manufactured by Fuji        Spinning Co., Ltd.)    -   Scale: 6 L of carrier, 8 L of wort    -   Strains Used: A-2 (Comparative Example 1), NA-3 (Example 3),        NA-4 (Example 4)    -   Yeast Immobilizing Method: Yeast is immobilized as being brought        into contact with the carrier for 2 days while being circulated        according to a conventional method (650 g of sludge yeast with        respect to 6 L of carrier).    -   Liquid Space Velocity: 6 to 12 cm/min    -   Fermentation temperature: constantly 8° C.    -   Fermentation Time: 48 hours/batch    -   Batch number of Fermentation times: 7    -   Wort Used: wort adjusted to yield a sugar index of 11% Plato.

Fermentative Property Test

In each primary fermentation process, the amount of consumption ofextract during 24 hours after starting fermentation determined by avibration type densitometer (DMA58, manufactured by Anton Paar GmbH) wasused as an index for fermentation rate comparison. Thus obtained resultsare shown in Table 3 and FIG. 3.

As can be seen from the results shown in Table 3 and FIG. 3, thefermentation rate converged to substantially a constant value (about6.7%/24 hr) at the first process and later in the case where thenon-flocculent strains were used. The primary fermentation was completedin about 2 days. When the non-flocculent strains were used, no yeast wasseen to precipitate in the reactor.

In the case where the flocculent strain was used, the flocculated andsedimented yeast tended to precipitate within the reactor vessel, sothat the amount of yeast involved in fermentation increased, therebyaccelerating the fermentation rate and making it inconstant. Here, thefermentation rate was slower in the third fermentation process than inthe second fermentation process in the case where the flocculent strainwas used, because of the fact that the precipitated yeast was removed byexchanging malts in a short period of time therebetween. Thus, thefermentation rate was hard to control when the flocculent strain wasused, and it was necessary to carry out processing such as eliminationof precipitated yeast in order to stabilize the fermentation rate.

TABLE 3 AMOUNT OF EXTRACT CON- SUMPTION IN 24 HR AFTER STARTINGFERMENTATION NUMBER OF FERMENTATION (%) TIMES 1 2 3 4 AVERAGE NA-3 6.47.0 7.2 6.8 6.8 NA-4 5.9 6.7 6.8 6.6 6.5 A-2*¹ 8.2 9.6 8.1 8.5 8.6 *¹InA-2, the inside of column was washed with wort (malt) between the secondand third processes, so as to control the fermentation rate.

Floating Yeast Cell Counting Test

After the end of each fermentation process, the number of floating yeastcells in the fermentation liquid was counted in the same manner asExample 1. Thus obtained results are shown in Table 4 and FIG. 4.

As can be seen from the results shown in Table 4 and FIG. 4, whennon-flocculent strains were used under the above-mentioned conditions,the number of floating yeast cells upon the end of fermentation wasstably held at high levels, i.e., 23 to 39 million cells/ml in NA-3, and18 to 33 million cells/ml in NA-4. When the flocculent strain was used,by contrast, the number was 5 to 31 million cells/ml, and yeast was seento sediment within the reactor in the last half of repeated batchfermentation, and the number of floating yeast cells upon the end offermentation greatly fluctuated under the influence thereof.

TABLE 4 NUMBER OF FLOATING YEAST CELLS UPON THE END OF PRIMARYFERMENTATION NUMBER OF FERMENTATION (10⁶ cells/ml) TIMES 1 2 3 4 AVERAGENA-3 24 23 39 28 28.5 NA-4 29 33 30 18 27.5 A-2 5 12 25 31 18.3

Diacetyl Generated Amount Test

After the end of each primary fermentation process, the amount ofgeneration of diacetyl (DA) in the fermentation liquid was measured by agas chromatography (GC-14B, manufactured by Shimadzu Corp.). Thusobtained results are shown in Table 5 and FIG. 5.

As can be seen from the results shown in Table 5 and FIG. 5, the amountof generation of diacetyl upon the end of primary fermentation wasstably maintained at a low level of 0.3 to 0.6 ppm when thenon-flocculent strains were used. When the flocculent strain was used,by contrast, the amount was 0.4 to 1.3 ppm. It was seen that the amountof generation of diacetyl when the non-flocculent strains were used wasabout half that when the flocculent strains were used and was stablyheld at this low level.

TABLE 5 AMOUNT OF DA UPON THE END OF PRIMARY FERMENTATION (ppm) NUMBEROF FERMENTATION TIMES 1 2 3 4 5 6 7 AVERAGE*² NA-3 0.42 0.36 0.36 0.330.60 0.38 0.41 0.41 NA-4 0.55 0.49 0.39 0.42 0.37 0.44 0.42 0.44 A-20.53 0.42 0.98 0.49 1.27 0.90 0.78 0.77 *²normal level: about 0.4 ppm

Example 5

Using the fluidized bed type bioreactor (total volume: 450 liters) shownin FIG. 1, the primary fermentation process was carried out by theabove-mentioned repeated batch fermentation (repeated batch type) underthe following conditions. Then, thus obtained fermentation liquid wassubjected to a fermentative property test, a floating yeast cellcounting test, and a diacetyl generated amount test by the same methodsas mentioned above. Further, in this example, a flavor component testand an organoleptic test were carried out for the final product.

Primary Fermentation Conditions

-   -   Carrier: chitosan type beads (Chitopearl HP manufactured by Fuji        Spinning Co., Ltd.)    -   Scale: 120 L of carrier, 170 L of wort    -   Strains Used: NA-5 (non-flocculent strain)    -   Yeast Immobilizing Method: About 13 kg of sludge yeast were        immobilized as being brought into contact with 120 L of the        carrier while being circulated according to a conventional        method as in Examples 3 and 4.    -   Liquid Space Velocity: 6 to 12 cm/min    -   Fermentation temperature: constantly 8° C.    -   Fermentation Time: 48 hours/batch    -   Batch number of Fermentation times: 9    -   Wort Used: wort adjusted to yield a sugar index of 11% Plato.

Secondary Fermentation Conditions Scale: 30 L Period: 4 weeks

Secondary Fermentation Temperature: 8 to 0° C.

In the fermentative property test of this example, as can be seen fromthe results shown in Table 6 and FIG. 6, the fermentation rate after thecompletion of adaptation period became substantially a constant value(average value of about 5.8%/24 hr). The precipitation of yeast withinthe bioreactor, which is seen when flocculent strains are used, was notobserved in this case as well.

TABLE 6 NUMBER OF FERMENTATION TIMES 1 2 3 4 5 6 7 8 9 AVERAGE AMOUNT OF5.6 5.8 5.4 5.8 5.3 6.2 5.7 6.7 6.0 5.8 EXTRACT CONSUMP- TION (%/24 HR)

In the floating yeast cell counting test of this example, as can be seenfrom the results shown in Table 7 and FIG. 7, the number was at a levelof 10 to 20 million cells/ml (with an average value of about 15 millioncells/ml), which was slightly lower than that in other examples usingother smaller scale bioreactors, but it was possible for the number tobe stably held at a level higher than a common level (3 to 10 millioncells/ml) of the case where the flocculent strain was used in the80-liter-scale bioreactor.

TABLE 7 NUMBER OF FERMENTATION TIMES 1 2 3 4 5 6 7 8 9 AVERAGE NUMBER OF14 17 17 21 13 10 11 11 18 14.6 FLOATING YEAST CELLS (× 10⁶ cells/ml)

In the diacetyl generated amount test in this example, as can be seenfrom the results shown in Table 8 and FIG. 8, the amount of diacetylgeneration upon the end of primary fermentation was 0.5 to 0.7 ppm (withan average value of about 5.8 ppm).

TABLE 8 NUMBER OF FERMENTATION TIMES 1 2 3 4 5 6 7 8 9 AVERAGE DA (ppm)0.50 0.48 0.50 0.54 0.67 0.52 0.52 0.50 0.64 0.54

The beer having completed the primary fermentation in the bioreactor wassubsequently subjected to secondary fermentation for 4 weeks at a scaleof 30 liters. Thereafter, analyses of low volatile components (L.V.C.)and diacetyl, and an organoleptic test were carried out for the finalproduct beer. The analysis of low volatile components was carried outwith a gas chromatography GC4A manufactured by Shimadzu Corp.

Table 9 shows the results of analyses of low volatile components anddiacetyl concerning the final product beer obtained by use of the beeryielded by the ninth primary fermentation. As can be seen from theresults shown in Table 9, the final product beer obtained by thisexample was characterized in that the amount of acetate was higher thanthe analyzed value of that in a standard beer, whereas the amount ofdiacetyl, which might become problematic in bioreactor fermentation inparticular, was reduced and lowered to 0.02 ppm, which was at a levelhardly detectable organoleptically.

The results of a drinking test for the final product beer obtained inthis example was not so different from those of conventional beers, andwere favorable in general.

TABLE 9 ANALYSIS RESULT (ppm) ACET- ETHYL ISOAMYL ALDEHYDE ACETONEACETATE n-PrOH i-BuOH ACETATE i-AmOH DA REACTOR 4.3 0.1 36 14.6 12.7 2.056 0.02 FERMENTATION BEER 1.4 0.6 20 9.2 7.1 1.5 50 0.01 STANDARD VALUE

INDUSTRIAL APPLICABILITY

According to the method of the present invention, as explained in theforegoing, non-flocculent yeast is used contrary to the conventionalknowledge when making a fermentation product by use of a bioreactorutilizing an immobilized microorganism, whereby the fermentation rate inthe fermentation process can be held constant, and the number offloating yeast cells upon the end of fermentation can stably bemaintained at a higher level.

When the method of the present invention is employed in the case ofmaking a malt alcohol beverage by use of a bioreactor in particular, theamount of diacetyl in the fermentation liquid and final product can belowered sufficiently and so forth, whereby the flavor of product can beimproved.

1. A method for making a fermentation product comprising: immobilizing anon-flocculent yeast in a bioreactor, contacting a fermentation liquidwith the immobilized non-flocculent yeast for a time and underconditions suitable for fermentation, and recovering the fermentationproduct(s), wherein said non-flocculent yeast is immobilized on acarrier selected from the group consisting of a chitosan bead, alginicacid and carrageenan and wherein said non-flocculent yeast satisfies thefollowing: a suspension of 0.6 g of said yeast is mixed with 20 ml ofwater, 1 ml of 0.5 M acetic acid buffer solution at pH 4.5 including1,500 ppm calcium ion is added to 9 ml of said suspension, the resultingmixture is left to stand still for 5 minutes at room temperature, andneither flocculation nor sedimentation of said yeast is observed.
 2. Themethod of claim 1, wherein said non-flocculent yeast is immobilized on achitosan bead.
 3. The method of claim 1, wherein said non-flocculentyeast is immobilized on alginic acid.
 4. The method of claim 1, whereinsaid non-flocculent yeast is immobilized on carrageenan.
 5. The methodof claim 1, further comprising subjecting the fermentation products to asecondary fermentation.
 6. The method of claim 1, wherein saidnon-flocculent yeast is a liquor yeast.
 7. The method of claim 1,wherein said non-flocculent yeast is a beer yeast.
 8. The method ofclaim 1, wherein said non-flocculent yeast is Saccharomyces cerevisiae.9. The method of claim 1, wherein said non-flocculent yeast isSaccharomyces uvarum.
 10. The method of claim 1, wherein said bioreactorcomprises a complete mixed vessel reactor.
 11. The method of claim 1,wherein said bioreactor comprises a packed bed reactor.
 12. The methodof claim 1, wherein said bioreactor comprises a film reactor.
 13. Themethod of claim 1, wherein said bioreactor comprises a fluidized bedreactor.
 14. The method of claim 1, wherein said bioreactor comprises alateral reactor.
 15. The method of claim 1, wherein said fermentationliquid comprises malt.
 16. The method of claim 1, wherein saidfermentation liquid comprises a fruit juice.
 17. The method of claim 1,wherein said fermentation liquid comprises a sugar liquid.
 18. Themethod of claim 1, wherein said fermentation liquid comprises a cerealsaccharified liquid.
 19. The method of claim 1, wherein said fermentedproduct is beer.
 20. The method of claim 1, wherein said fermentedproduct is a malt alcohol beverage.
 21. The method of claim 1, whereinsaid fermented product is sake.
 22. The method of claim 1, wherein saidfermented product is wine.
 23. The method of claim 1, wherein saidfermented product is vinegar.
 24. The method of claim 1, wherein saidfermented product is soy sauce.
 25. A fermentation method comprising:immobilizing a non-flocculent yeast on an immobilizing carrier within abioreactor having a fluidized bed section, supplying the bioreactor witha fermentation liquid, extracting a part of the fermentation liquid fromthe downstream side of the fluidized bed section and returning the partof the fermentation liquid to the upstream side of the fluidized bedsection, while forming a fluidized bed to carry out the fermentation ofthe fermentation liquid; and recovering the thus obtained fermentationproduct from the bioreactor and optionally supplying the bioreactor witha new fermentation liquid to repeat the fermentation, wherein saidnon-flocculent yeast satisfies the following: a suspension of 0.6 g ofsaid yeast is mixed with 20 ml of water, 1 ml of 0.5 M acetic acidbuffer solution at pH 4.5 including 1,500 ppm calcium ion is added to 9ml of said suspension, the resulting mixture is left to stand still for5 minutes at room temperature, and neither flocculation norsedimentation of said yeast is observed.
 26. The method of claim 25,wherein said non-flocculent yeast is immobilized on a carrier selectedfrom the group consisting of a chitosan bead, alginic acid andcarrageenan.
 27. The method of claim 25, wherein the immobilizingcarrier is a chitosan bead.
 28. The method of claim 25, wherein thenon-flocculent yeast is a non-flocculent liquor yeast and thefermentation product is a liquor.
 29. The method of claim 25, whereinthe non-flocculent yeast is a non-flocculent bear yeast and thefermentation product is a malt alcohol beverage.
 30. A method for makinga fermentation product comprising: immobilizing a non-flocculent yeastin a fluidized bed bioreactor on the surface of a chitosan bead,contacting a fermentation liquid with the immobilized non-flocculentyeast for a time and under conditions suitable for fermentation,recovering the primary fermentation product(s), wherein the primaryfermentation product(s) have a higher number of floating yeast cellsthat the primary fermentation product(s) obtained using a similarflocculent yeast of the same species, and wherein said non-flocculentyeast satisfies the following: a suspension of 0.6 g of said yeast ismixed with 20 ml of water, 1 ml of 0.5 M acetic acid buffer solution atpH 4.5 including 1,500 ppm calcium ion is added to 9 ml of saidsuspension, the resulting mixture is left to stand still for 5 minutesat room temperature, and neither flocculation nor sedimentation of saidyeast is observed.